Beginning with this chapter we'll be sticking somewhat closer to the textbook in terms of the material. The good thing about that is that you don't need to rely heavily on these lecture notes; the bad thing (perhaps) is that you'll be responsible for everything that is in the textbook; so if there's something in Horton that you don't understand, then check out the web resources for the course or ask for help from me or one of the other biochemists here at IIT.
We want to understand the atomic-level events that take place in the course of an enzymatically-catalyzed reaction. In some cases we want to know these things because we want to find a way to inhibit an enzyme; in other cases we're looking for more fundamental knowledge, viz. the ways that biological organisms employ chemistry and how enzymes make that chemistry possible. There are a variety of experimental tools available for understanding mechanisms, including isotopic labeling of substrates, structural methods, and spectroscopic kinetic techniques.
Many of the reactions we'll be studying are ionic; that is, they involve ionic intermediates. Typically two species are involved: one is electron-rich, and the other is electron-poor. These species are known as a nucleophile and an electrophile respectively. We conventionally discuss the reaction as an attack by the nucleophile on the electrophile, and we conventionally draw this attack by showing the nucleophile donating its electron to the electrophile, as shown in schemes 6.1 and 6.2 in Horton.
The reactions may be substitutions, as in these two figures. These depict transition states in which the nucleophile becomes covalently bonded to the electrophile, and then eventually a leaving group (itself a nucleophile, but presumably less energetic) goes off from the transition state. We make a distinction in biochemistry between a transition state, which is a short-lived, high-energy species, detectable only by rapid spectroscopic techniques; and an intermediate, which is a longer-lived state in between reactants and products. Figure 6.2 illustrates the difference between a transition state and an intermediate on a free-energy diagram.
Other enzymatically catalyzed reactions involve cleavages, in which a carbanion or carbocation appears in the transition state. All of the ionic species we see in nucleophilic substitution and cleavage reactions involve even numbers of electrons: that is, every neutral and charged species has an even number of electrons in it. There are some enzymatic reactions (such as that involving superoxide, discussed later in this chapter) in which a free radical, i.e. a species containing an odd number of electrons, is involved. Surveillance of free radicals, which tend to be indiscriminately and strongly reactive, is an important biological function, and we will encounter it numerous times in this course. Free radicals may be charged or uncharged; the species shown in scheme 6.5 in the text are uncharged, but charged radical species, like superoxide, O2-., do appear in numerous biological reactions. Water can readily dissociate under the influence of ionizing radiation into uncharged radicals, H. and OH., and these can undergo further reactions to produce both charged and uncharged radicals.
Oxidation-reduction reactions are commonplace in biochemistry as well,
as indicated by the fact that Enzyme Commission Class I enzymes
are the oxidoreductases.
Typically the organic reactants in oxidation-reduction reactions
experience an increase or decrease in the number of C-H bonds:
thus, an primary alcohol (R-CH2-OH) is converted to an
aldehyde (R-CH=O), so that the alcohol is being oxidized; some other
compound (often NADP in the short run, but ultimately molecular
oxygen) is being reduced. Thus the net reaction is
R-CH2-OH + (1/2)O2 -> R-CH=O + H2O
Similarly, the conversion of secondary alcohol to a ketone, an aldehyde to a carboxylic acid, or an amine group to a species containing a C=N bond involve oxidation of the reactant. All other things being equal, reduced compounds are usually somewhat higher-energy than oxidized compounds, but these reactions can be reversible, given appropriate coupling to exergonic reactions.
Fundamentally, the job of an enzyme is to stabilize the transition state partway between reactants and products so that the energy required to get from one side of the reaction to the other is small. We have been using energy diagrams for several weeks to depict this process; figs. 6.1 and 6.2 in Horton should be familiar by now. The energy associated with overcoming the barrier between reactants and products is typically denoted as ΔG. Thus the job of an enzyme is to reduce ΔG.
One aspect of activation energy that is not discussed in the
book is how to measure it. A simple method is to examine the temperature
dependence of a reaction. Suppose for simplicity that the
for a reaction is zero, i.e. that the energies of the reactants and
products are equal. Then the temperature dependence of the rate of the
reaction will follow this simple relationship:
k(T) = Q0exp(-ΔG/kT)
where the k on the right side of the equation is Boltzmann's constant, T is the Kelvin temperature, and Q0 is a normalization factor. Thus a plot of ln(k(T)) against 1/T will have a straight line with slope equal to -ΔG/k, and we can deduce the value of ΔG from that.
Enzymes reduce ΔG by allowing the binding of the transition state into the active site; in fact, the binding of the transition state needs to be tighter than the binding of either the reactants or the products.
The effect is partly entropic: when a substrate binds, it loses a lot of entropy. Thus the entropic disadvantage of (say) a bimolecular reaction is soaked up in the process of binding the first of the two substrates into the enzyme's active site. But there is often an enthalpic component to the reduction in ΔG as well; ionic or hydrophobic interactions between the enzyme's active site residues and the components of the transition state make that transition state more stable. Fig. 6.3 shows the influences of various aspects of catalysis on reaction rates.
What are the actual chemical reactions that happen in enzymatic systems? Many involve changes in the charge states of various amino acid side chains and (rarely) the terminal amino and carboxyl groups of the protein. Table 6.1 lists the charge states at pH=7 of some common amino acid side chains. Note that the pKa of an amino acid within a protein may be distinctly different from its solution value; that accounts for the discrepancies between table 6.1 and table 3.2. Histidine is often an acceptor or donor of protons; asp, glu, and lys can be involved in proton transfer as well. Ser and cys can become involved in group transfers, including hydrolysis of peptide or ester bonds.
Acid-base catalysis involves speeding up a reaction involving the net transfer of a proton from one species to another. Schemes 6.7 and 6.8 in Horton illustrate general base (B:) catalysis; scheme 6.9 illustrates general acid (BH+) chemistry.
Many reactions involve a catalytic interaction between a side chain residue in a protein and a substrate. Schemes 6.10 and 6.11 describe participation of enzymes in these kinds of reactions. Note that a charged residue may participate in a reaction without changing its nominal protonation state, i.e. without forming or breaking any of its own covalent bonds. A carboxylate (from an aspartate or glutamate side-chain, or from the carboxyl terminus of the protein) in the vicinity of a positively charged group in a reaction intermediate can stabilize that positive charge, thus lowering the enthalpy of the positively charged group. Similarly, a positvely charged group (from a lysine or arginine side-chain or from the amino terminus) can stabilize a negative charge on an intermediate in its vicinity. So protonation and deprotonation are not the only circumstances where a charge in an active site can play a role in a reaction mechanism. Many enzymatic active sites are fairly hydrophobic, so the presence of a charge in the active site comes at an energetic price; but that price is worth paying if the charged residue can stabilize a transition state.and deprotonation are not the only circumstances where a charge in an active site can play a role in a reaction mechanism. Many enzymatic active sites are fairly hydrophobic, so the presence of a charge in the active site comes at an energetic price; but that price is worth paying if the charged residue can stabilize a transition state. We can determine whether changes in charge state are important to a reaction by examining the effect of pH on the enzymatically-catalyzed reaction rate. If there is a significant change in reaction rate near the pKa of a side-chain, then it is likely that that amino acid is involved in catalysis. Fig. 6.4 illustrates this point.
Catalysis by a general base is illustrated in scheme 6.7, in which a general base (shown as :B in the scheme) donates an electron pair to extract a hydrogen from an electrophile, converting the base to its positively-charged conjugate acid and leaving a negative charge on the former electrophile. Catalysis by a general base is shown in scheme 6.8 for cleavage of an amide (peptide) bond; here the intermediate is tetrahedral, and involves a carbon atom with two oxygens and a nitrogen bonded to it. The outcome of the reaction is (formally) a carboxylic acid and an amine; in practice a carboxylate ion and an substituted ammonium ion (NH4+) will appear.
General acids can participate in cleaving bonds as well, as illustrated in scheme 6.9. Here the starting material is the amine, R-OH; the R-O bond cannot be readily cleaved to produce an ion pair, but in the presence of a proton donor (a general acid), the formation of an unstable intermediate, R-OH2+ leads to cleavage of the R-O bond to produce a carbocation and water.
Often the enzyme's participation in a reaction involves formation
of a covalent intermediate, as given in schemes 6.10 and 6.11:
A-X + E = X-E + A
X-E + B = B-X + E
Here the enzyme begins and ends the reaction pathway as free (uncomplexed) enzyme; but in the middle, the leaving group X that had been attached to the starging material A ends up covalently attached to E, only to be abstracted from there and attached to the second reactant B. This kind of mechanism is by definition sequential, since the intermediate X-E must be present before the product B-X can form. Horton offers an example from carbohydrate chemistry, in which the overall reaction is
sucrose + Pi = glucose-1-phosphate + fructose
but the ability to produce G-1-P depends on a a covalent glucosyl derivative of the enzyme and free fructose. This glucosyl derivative can be phosphorylated to produce the proper final product and the reconstituted, free enzyme.